In sexually reproducing species, gametes are the only cells in the body that can give rise to a genetically complete new organism. In this sense, "gametes are the stem cells of the species" (Wylie, 1999). Gametes originate from a small population of cells called primordial germ cells (PGCs) that form early in development. These unique cells retain full totipotency while somatic cells become progressively restricted in their fates. Eventually, PGCs migrate from their site of origin to the somatic gonads where they divide, enter meiosis and sexually differentiate. In Xenopus, it is thought that PGCs are determined by inheritance of germ plasm, a specialized cytoplasmic domain containing unique RNAs and proteins. Virtually nothing is known about how PGCs preserve their totipotency or what functions germ plasm components play in POC development. Several germ plasm specific RNAs have been isolated, including Xdazl a member of the DAZ family in humans (King et al., 1999). We found that maternal depletion of Xdazl results in the severe reduction or loss of PGCs because they fail to migrate out of the endoderm (Houston and King, 2000). Xcat-2, DEADSouth, and Xpat are other germ plasm specific RNAs that have been characterized, but whose functions are not known. In this proposal, experiments are outlined to explore how germ cell fate is determined and will address the following questions: I. What mechanism permits PGCs to retain a totipotent fate while surrounding cells in the vegetal mass become committed to an endoderm fate? What role, if any, does endoderm play in this process? II. What steps in PGC development require the germ plasm components Xcat-2, DEADSouth, Xdazl, and Xpat? What specific molecular functions do Xdazl and DEADSouth proteins perform? III. What is the mechanism of translational repression of the germ plasm RNAs Xdazl, Xcat2, and DEADSouth during oogenesis? A method for targeting green fluorescent protein (GFP) expression to the germ plasm is presented that will allow PGCs to be followed or isolated at different times in development. This procedure will make PGCs accessible to biochemical and molecular studies for the first time. Immunocytochemistry and in situ hybridization will be used to determine when PGCs become transcriptionally active and whether they express genes diagnostic for endoderm. Gain-of-function, loss-of-function, and over-expression experiments targeting the germ plasm RNAs will pinpoint the steps that require these genes. Oocyte injections of deletion mutants in the UTR regions of Xcat2 and Xdazl will reveal cis-acting elements required for translational repression. These cis elements will be used to affinity purify trans-acting factors. Results from these proposed studies will offer significant insights into how the germline forms in Xenopus and in general as key aspects of PGC development (totipotency, migration) are likely conserved across phyla.